Microbial production of indigo

ABSTRACT

There is provided an improved process for the biosynthetic production of indigo, the improvement comprising removing unwanted by-products such as isatin or indirubin from the broth in which such indigo is produced. Isatin can be removed by enzymatic activity using an isatin-removing enzyme such as an isatin hydrolase, or by other techniques such as process parameters (elevated temperature, pH), or by contacting the broth containing the isatin with appropriate adsorption compounds/compositions such as carbon or appropriate resins. Since isatin is the precursor of indirubin, the indirubin levels are decreased as a result of isatin removal.

This application is a continuation of Ser. No. 08/560,729 Nov. 20, 1995U.S. Pat. No. 6,190,892.

FIELD OF THE INVENTION

The present invention relates to the biosynthetic production of dyestuff, and particularly indigo, from microorganisms. While thebiosynthetic production of indole and tryptophan (both precursors toindigo) have been previously described, the present invention providesan efficient, commercially feasible biosynthetic production systemwhereby an inhibitory compound in the synthesis of indigo, isatin, isreduced or otherwise removed from fermentation broth. The removal ofthis inhibitory compound during production of indigo provides enhancedproduction of the desired end product indigo and/or improvedcharacteristics of the indigo so produced. Furthermore, the presentinvention prevents or reduces the production of indirubin, a reddyestuff which is a by-product of biosynthetic indigo production.

BACKGROUND OF THE INVENTION

The blue dye indigo is one of the oldest dyestuffs known to man. Its useas a textile dye dates back to at least 2000 BC. Until the late 1800sindigo, or indigotin, was principally obtained from plants of the genusIndigofera, which range widely in Africa, Asia, the East Indies andSouth America. As the industrial revolution swept through Europe andNorth America in the 1800s, demand for the dye's brilliant blue colorlead to its development as one of the main articles of trade betweenEurope and the Far East. In 1883 Alfred von Baeyer identified theformula of indigo: C₁₆H₁₀N₂O₂. In 1887 the first commercial chemicalmanufacturing process for indigo was developed. This process, still inuse today, involves the fusion of sodium phenylglycinate in a mixture ofcaustic soda and sodamide to produce indoxyl. The process' finalproduct, indoxyl, oxidized spontaneously to indigo by exposure to air.

Current commercial chemical processes for manufacturing indigo result inthe generation of significant quantities of toxic waste products.Obviously, a method whereby indigo may be produced without thegeneration of toxic by-products is desirable. One such method whichresults in less toxic by-product generation involves indigo biosynthesisby microorganisms.

Ensley et al. [(1983) Science 222:167-169] found that a DNA fragmentfrom a transmissible plasmid isolated from the soil bacteriumPseudomonas putida enabled Escherichia coli stably transformed with aplasmid harboring the fragment to synthesize indigo in the presence ofindole or tryptophan. Ensley et al. postulated that indole, added eitheras a media supplement or produced as a result of enzymatic tryptophancatabolism, was converted to cis-indole-2,3-dihydrodiol and indoxyl bythe previously identified multi-component enzyme naphthalene dioxygenase(NDO) encoded by the P. putida DNA fragment. The indoxyl so produced wasthen oxidized to indigo upon exposure to air. The dioxygenase describedby Ensley et al. is a preferred oxygenase useful in the production ofindigo as further described herein.

NDO had previously been found to catalyze the oxidation of the aromatichydrocarbon naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene [Ensley et al. (1982) J.Bacteriol. 149:948-954]. U.S. Pat. No. 4,520,103, incorporated byreference, describes the microbial production of indigo from indole byan aromatic dioxygenase enzyme such as NDO. The NDO enzyme is comprisedof multiple components: a reductase polypeptide (Rd, molecular weight ofapproximately 37,000 daltons (37 kD)); an iron-sulfur ferredoxinpolypeptide (Fd, molecular weight of approximately 13 kD); and aterminal oxygenase iron-sulfur protein (ISP). ISP itself is comprised offour subunits having an α₂β₂ subunit structure (approximate subunitmolecular weights: α, 55 kD; β,21 kD). ISP is known to bind naphthalene,and in the presence of NADH, Rd, Fd and oxygen, to oxidize it tocis-naphthalene-dihydrodiol. Fd is believed to be the rate-limitingpolypeptide in this naphthalene oxidation catalysis, (see U.S. Pat. No.5,173,425, incorporated herein by reference, for a thorough discussionof the various NDO subunits and ways to improve them for purposes ofindigo biosynthesis).

In addition, aromatic dioxygenases other than NDO may also be useful inthe biosynthetic production of indigo, for example, a toluenemonooxygenase (TMO) such as that from Pseudomonas (P. mendocina) capableof degrading toluene was also able to produce indigo when the culturemedium was supplemented with indole. For details, see U.S. Pat. No.5,017,495, incorporated herein by reference. In principle, any enzymecapable of introducing a hydroxyl moiety into the 3-position of indoleto give indoxyl is a candidate for use in the biosynthetic production ofindigo.

It has also long been known that microorganisms contain biosyntheticpathways for the production of all 20 essential amino acids, includingthe aromatic amino acid L-tryptophan. The de novo synthesis of aromaticamino acids (phenylalanine, tryptophan and tyrosine) share a commonpathway up through the formation of chorismic acid. After chorismic acidsynthesis, specific pathways for each of the three aromatic amino acidsare employed to complete their synthesis.

Bacterial biosynthesis of tryptophan from chorismic acid (specificallyin E. coli) is under the control of the tryptophan (trp) operon. The(trp) operon, comprised of regulatory regions and several structuralgenes, has been extensively studied because of its complex andcoordinated regulatory systems. The regulatory and structuralorganization of the E coli trp operon, along with the catalyticactivities encoded by the structural genes of the operon, appear in FIG.1 of PCT/US93/09433, incorporated herein by reference. PCT/US93/09433describes improvements in the intracellular production of indole,specifically as it relates to the conversion ofindole-3′-glycerol-phosphate (InGP), in conjunction with L-serine, toL-tryptophan. The reaction is catalyzed by the multi-subunit enzymetryptophan synthase. During the reaction, indole is produced as anintermediate.

However, the indole is very rapidly combined with L-serine in astoichiometric fashion to produce L-tryptophan. Thus, no free indole isproduced as a result of this InGP plus L-serine conversion totryptophan.

However, Yanofsky et al. [(1958) Biochim. Biophys. Acta. 28:640-641]identified a tryptophan synthase mutant which led to the accumulation ofindole. This particular tryptophan synthase mutant, however, was subjectto spontaneous reversion to the wild-type phenotype, as the mutationresulted from a single nucleotide base pair change in the gene codingfor the β subunit of tryptophan synthase.

PCT/US93/09433 describes a method for creating stable tryptophansynthase mutants capable of yielding high levels of intracellularindole. When such indole accumulating tryptophan synthase mutantsexpress an aromatic dioxygenase enzyme like NDO, the accumulated indolemay be converted to indoxyl, which can then be oxidized to indigo bymolecular oxygen. Thus, through the commercial application ofrecombinant DNA technology, by the overexpression of a modified trpoperon capable of continuously producing indole and an oxygenase enzymecapable of simultaneous conversion of indole to indoxyl, indigo can beproduced from a renewable raw material such as glucose.

In shake flask studies applicants have determined that during thesynthesis of indigo from indole, low levels of other compounds orby-products accumulate in the culture supernatant. One of theseby-products, isatin (indole 2,3-dione), has been found to inhibit theoxygenase (i.e., NDO) activity in the production strain and,consequently, reduces overall indigo production; thus, isatin isundesirable. In addition to the by-product isatin, indirubin, a red dyematerial derived from isatin, may be produced during this biosyntheticindigo production process. The by-product isatin is believed to reducethe productivity of the production strain, while the by-productindirubin is believed to cause undesirable dyeing characteristics tomicrobially produced indigo which is expressed as a red cast on clothdyed with indirubin-tainted microbially produced indigo.

Because the production in shake flasks of one or more of theseby-products may either reduce the productivity of this production strainand/or cause undesirable characteristics of the indigo producedtherefrom, an object of the present invention is to reduce the buildupof isatin or remove such isatin formed as a by-product of biosynthesisof indigo in microbial cells. Removal of isatin will potentially enhancethe overall production of indigo in a fermentor and reduce or preventthe accumulation of indirubin. One method to reduce the buildup ofisatin or remove such isatin, as detailed herein, relates to theisolation, cloning, sequencing and expression in indigo-producing hoststrains of a gene encoding an enzyme having isatin-removing activity.Preferably the enzyme is an isatin hydrolase, an enzyme capable ofdegrading isatin; however, any method to remove or inhibit isatinformation is contemplated by the present invention. Thus, another aspectof the present invention is the enhanced production of biosyntheticindigo by reducing the buildup or removing accumulated isatin throughmeans, including, but not limited to, enzymatic conversion of the isatinby contacting it with an isatin-removing enzyme such as an isatinhydrolase, general base catalyzed chemical conversion of the isatin atappropriate temperature and pH, or through adsorption of the isatin tocarbon or a suitable resin. These aspects of the invention are detailedbelow.

Definition of Terms

The following terms will be understood as defined herein unlessotherwise stated. Such definitions include without recitation thosemeanings associated with these terms known to those skilled in the art.

Tryptophan pathway genes useful in securing biosynthetic indoleaccumulation include a trp operon, isolated from a microorganism as apurified DNA molecule that encodes an enzymatic pathway capable ofdirecting the biosynthesis of L-tryptophan from chorismic acid. (A.J.Pittard (1987) Biosynthesis of Aromatic Amino Acids in Escherichia coliand Salmonella typhimurium, F. C. Neidhardt, ed., American Society forMicrobiology, publisher, pp. 368-394.) Indole accumulation is enabled bymodification of one or more of the pathway's structural elements and/orregulatory regions. This modified trp operon may then be introduced intoa suitable host such as a microorganism, plant tissue culture system orother suitable expression system. It should be noted that the term“indole accumulation” does not necessarily indicate that indole actuallyaccumulates intracellularly. Instead, this term can indicate that thereis an increased flux of carbon to indole and indole is made available asa substrate for intracellular catalytic reactions such as indoxylformation and other than the formation of L-tryptophan. In the contextof this invention, the “accumulated” indole may be consumed in theconversion of indole to indoxyl by an oxygenase such as the aromaticdioxygenase NDO, or an aromatic monooxygenase such as TMO, or it mayactually build up intracellularly and extracellularly, as would be thecase when the desired end product is indole or one of its derivatives.

A suitable host microorganism or host cell is an autonomoussingle-celled organism useful for microbial indole and/or indigoproduction and includes both eucaryotic and procaryotic microorganisms.Such host microorganism contains all DNA, either endogenous orexogenous, required for the production of indole, indoxyl and/or indigo,either from glucose or as a bioconversion from tryptophan. Usefuleucaryotes include organisms like yeast and fungi or plants. Prokaryotesuseful in the present invention include, but are not limited to,bacteria such as E. coli, P. putida and Salmonella typhimurium.

Biosynthetic conversion of indole to indigo is meant to include indoxyloxidation to indigo mediated by molecular oxygen or air.

A DNA fragment, as used herein, may encode regulatory and/or structuralgenetic information. A DNA fragment useful in the instant inventionshall also include: nucleic acid molecules encoding sequencescomplementary to those provided; nucleic acid molecules (DNA or RNA)which hybridize under stringent conditions to those molecules that areprovided; or those nucleic acid molecules that, but for the degeneracyof the genetic code, would hybridize to the molecules provided or theircomplementary strands. “Stringent” hybridization conditions are thosethat minimize formation of double stranded nucleic acid hybrids fromnon-complementary or mismatched single stranded nucleic acids. Inaddition, hybridization stringency may be effected by the variouscomponents of the hybridization reaction, including salt concentration,the presence or absence of formamide, the nucleotide composition of thenucleic acid molecules, etc. The nucleic acid molecules useful in thepresent invention may be either naturally or synthetically derived.

An “exogenous” DNA fragment is one that has been introduced into thehost microorganism by any process such as transformation, transfection,transduction, conjugation, electroporation, etc. Additionally, it shouldbe noted that it is possible that the host cell into which the“exogenous” DNA fragment has been inserted may itself also naturallyharbor molecules encoding the same or similar sequences. For example,when E. coli is used in this invention as the host strain, it isrecognized that normally the host naturally contains, on its chromosome,a trp operon capable of directing the synthesis of L-tryptophan fromchorismic acid under conditions enabling trp operon expression. Amolecule such as this is referred to as an “endogenous” DNA molecule.

A stably transformed microorganism is one that has had one or moreexogenous DNA fragments introduced such that the introduced moleculesare maintained, replicated and segregated in a growing culture. Stabletransformation may be due to multiple or single chromosomalintegration(s) or by extrachromosomal element(s) such as a plasmidvector(s). A plasmid vector is capable of directing the expression ofpolypeptides encoded by particular DNA fragments. Expression may beconstitutive or regulated by inducible (or repressible) promoters thatenable high levels of transcription of functionally associated DNAfragments encoding specific polypeptides such as the structural genes ofa trp operon modified as described herein.

An “isatin-removing enzyme,” as used herein, is any enzyme whichcomprises activity resulting in the inhibition, removal, inactivation,degradation, hydrolysis or binding (sequestering) of isatin, whethersuch enzyme causes the formation of isatic acid or any other derivativeof isatin. A preferred isatin-removing enzyme useful in the presentinvention is an isatin hydrolase such as the hydrolase isolated fromPseudomonas putida (WW2) herein.

Regardless of the exact mechanism utilized for expression of enzymesnecessary for the microbial production of indole, indoxyl and/or indigo,it is contemplated that such expression will typically be effected ormediated by the transfer of recombinant genetic elements into the hostcell. Genetic elements as herein defined include nucleic acids(generally DNA or RNA) having expressible coding sequences for productssuch as proteins, specifically enzymes, apoproteins or antisense RNA,which express or regulate expression of relevant enzymes (i.e., isatinhydrolase, tryptophan synthase, NDO, etc.). The expressed proteins canfunction as enzymes, repress or derepress enzyme activity or controlexpression of enzymes. Recombinant DNA encoding these expressiblesequences can be either chromosomal (integrated into the host cellchromosome by, for example, homologous recombination) orextrachromosomal (for example, carried by one or more plasmids, cosmidsand other vectors capable of effecting the targeted transformation). Itis understood that the recombinant DNA utilized for transforming thehost cell in accordance with this invention can include, in addition tostructural genes and transcription factors, expression controlsequences, including promoters, repressors and enhancers, that act tocontrol expression or derepression of coding sequences for proteins,apoproteins or antisense RNA. For example, such control sequences can beinserted into wild-type host cells to promote overexpression of selectedenzymes already encoded in the host cell genome, or alternatively theycan be used to control synthesis of extrachromosomally encoded enzymes.

The recombinant DNA can be introduced into the host cell by any means,including, but not limited to, plasmids, cosmids, phages, yeastartificial chromosomes or other vectors that mediate transfer of geneticelements into a host cell. These vectors can include an origin orreplication, along with cis-acting control elements that controlreplication of the vector and the genetic elements carried by thevector. Selectable markers can be present n the vector to aid in theidentification of host cells into which genetic elements have beenintroduced. Exemplary of such selectable markers are genes that conferresistance to particular antibiotics such as tetracycline, ampicillin,chloramphenicol, kanamycin or neomycin.

A means for introducing genetic elements into a host cell utilizes anextrachromosomal multi-copy plasmid vector into which genetic elementsin accordance with the present invention have been inserted. Plasmidborne introduction of the genetic element into host cells involves aninitial cleaving of a plasmid vector with a restriction enzyme, followedby ligation of the plasmid and genetic elements encoding for thetargeted enzyme species in accordance with the invention. Uponrecircularization of the ligated recombinant plasmid, infection (e.g.,packaging in phage lambda) or other mechanism for plasmid transfer(e.g., electroporation, microinjection, etc.) is utilized to transferthe plasmid into the host cell. Plasmids suitable for insertion ofgenetic elements into the host cell are well known to the skilledartisan.

SUMMARY OF THE INVENTION

One aspect of the present invention is the isolation of an organismhaving an enzymatic activity for removing isatin (designated anisatin-removing enzyme), along with the cloning and sequencing of thegene encoding a preferred isatin-removing enzyme, isatin hydrolase.

Another aspect of the present invention is to incorporate DNA moleculesencoding isatin-removing enzymatic activity into host strains capable ofproducing indole, indoxyl and/or indigo. The DNA molecules arepreferably stably transformed, transfected or integrated into thechromosome of a procaryotic or eucaryotic host cell. Useful host cellsmay be bacteria, yeast or fungi, including, for example, Streptomyces,Escherichia, Bacillus, Pseudomonas, Saccharomyces, Aspergillus, etc. Theprocaryotic host Escherichia coli represents one preferred hostorganism.

A biologically functional plasmid or viral DNA vector, including a DNAmolecule of the invention, represents another aspect of the invention.In one embodiment, a eucaryotic or prokaryotic host cell such as E. coliis stably transformed or transfected with such a biologically functionalvector.

Other aspects of the invention involve methods for the biosynthesis ofindigo in a suitable host microorganism, the method comprisingintroducing into the host a DNA fragment encoding isatin-removing enzymeactivity and cultivating the microorganism under conditions facilitatingthe accumulation of indoxyl such that upon the conversion of indoxyl toindigo, the isatin-removing enzyme activity removes any isatinby-product produced. Suitable host microorganisms include, but are notlimited to, host organism(s) expressing (either endogenously orexogenously) the tryptophan operon (or a modified trp operon) and/oroxygenase activity, which host organism is stably transformed andtransfected with a DNA molecule encoding isatin hydrolase. Suchorganisms are cultivated under conditions facilitating the expression ofthe tryptophan operon (or modified trp operon), indole oxidizingactivity (to allow the formation of indoxyl) and the isatin hydrolase.

Specifically claimed is an improved process for the biosynthesis ofindigo in a selected host microorganism comprising introducing into thehost microorganism a DNA fragment encoding isatin-removing enzymeactivity capable of removing any isatin accumulated during theproduction of indigo, provided that the host microorganism can befurther modified by introducing one or more DNA fragments encoding oneor more of the following enzymatic activities:

(i) tryptophanase activity (capable of converting tryptophan to indole)or

(ii) oxygenase activity capable of converting indole to indoxyl; andcultivating the modified microorganism under conditions facilitatingexpression of polypeptides encoded by such DNA fragments such thatexpression of such polypeptides enables indole accumulation, conversionof indole to indoxyl and removal of isatin. Such modified microorganismswill allow the production of indoxyl, which is oxidized to indigo, andthe indigo so produced can then be recovered by means known to thoseskilled in the art.

Still another aspect of the present invention comprises an improvedmethod for making indigo whereby the production of the by-productsisatin and/or indirubin are inhibited or removed by any method,including chemical or enzymatic inhibition/inactivation or adsorptionwith compounds such as carbon.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the diagrammatic methodology used to clone the isatinhydrolase gene from P. putida strain WW2.

FIG. 2 shows the methodology used to subclone the isatin hydrolase genefrom P. putida strain WW2.

FIGS. 3A-3F shows the nucleotide sequence of the isatin hydrolase gene(Seq ID No. 1) and the deduced amino acid sequence (Seq ID No. 2), alongwith the 5′ and 3′ untranslated sequence (Seq. ID No. 1 1-144 bp and924-1006 bp, respectively) and polylinker sequence of the cloningvector.

FIG. 4 shows the construction of plasmid pUC-IH-H comprising the isatinhydrolase (IH) and tryptophanase (tnaA) genes.

FIG. 5 shows the construction of plasmid pCL-IH-SI comprising the isatinhydrolase gene in the same orientation as the lac promoter.

FIG. 6 shows the construction of plasmid pAK1, an intermediate plasmid.

FIG. 7 shows the construction of plasmid pCL-ISTI comprising the isatinhydrolase gene in the same orientation as the lac promoter and thetryptophanase (tnaA) gene.

FIG. 8 shows the construction of plasmid pCL-IHA comprising the isatinhydrolase gene in the same orientation as the lac promoter.

FIG. 9 shows the effect on bleaching of indigo-dyed denim due toaddition of indirubin to dye.

DETAILED DESCRIPTION OF THE INVENTION

Presently available methods of biosynthetic indigo production may employthe bioconversion of indole to indigo utilizing an aromatic mono- ordioxygenase like TMO or NDO, respectively, or other oxygenase enzymessuch as an oxidase from Rhodococcus, as described by S. Hart, K. R. Kochand D. R. Woods [(1992) “Identification of indigo-related pigmentsproduced by Escherichia coli containing a cloned Rhodococcus gene,” J.Gen. Microbiology138:211-216]. These processes necessitate the additionof indole to the culture medium, as no intracellular indole accumulationoccurs in such systems. However, indole added to the culture medium maybe toxic to microorganisms. E. coli growth may be inhibited when indoleis present in media. Bang et al. [(1983) Biotechnology andBioengineering25:999-101 1] described the effects of adding exogenousindole to E. coli is being grown in shake flasks in minimal media. Theyfound that while concentrations of up to 0.025% slowed bacterial growth,the cells acclimated to the presence of indole over time. However, 0.03%indole severely limited growth with no apparent acclimation, and indoleconcentrations above 0.04% prohibited growth altogether. In addition,Bang et al., supra, found that L-tryptophan synthesis was inhibited whenindole was added at concentrations in excess of 0.2 g/L.

To avoid the inherent limitations of indigo synthesis due to the needfor media supplementation with indole, systems capable of endogenousindole biosynthesis have been developed. One such system may employtransferring an exogenous DNA molecule encoding a DNA sequence for a trpoperon, modified so as to promote indole production and accumulation(hereinafter “modified trp operon”), into a recombinant hostmicroorganism already capable of expressing an oxygenase (i.e., NDO)activity. Such a system would allow for the production of indigo fromglucose or other carbon sources. Optimally, such a system wouldefficiently convert the endogenously produced indole to indoxyl in amanner avoiding intracellular indole accumulation. A detaileddescription of this system is found in PCT/US93/09433, incorporatedherein. Such a system in combination with the isatin-removing methods ofthe present invention is a preferred embodiment of the improved processfor indigo production described herein.

Generally, as shown in PCT/US93/09433, certain point mutations in trpBgenes, particularly at position 382 in the trpB gene (designatedtrpB382), resulted in stable mutants of the trpB. Such mutants lead toenhanced indole flux in the host cells and such mutants are preferredfor the production of indigo as described herein in combination withisatin-removing activity. Furthermore, expression of these tryptophansynthase mutants in conjunction with an aromatic dioxygenase such as NDOresulted in the intracellular production of indole and its conversion toindoxyl, which spontaneously oxidized to indigo.

The preferred embodiment of the present invention builds on theteachings of this prior work by addressing the problem of needing toeliminate or reduce the production of the undesirable by-products isatinand/or indirubin during microbial indigo production. Detailed below arespecifics regarding the enzymatic, chemical or adsorption methodsdeveloped to overcome this problem faced by applicants in their attemptsto scale up the biosynthetic production of indigo.

I. Enzymatic Approach to Isatin Formation Cloning of the IsatinHydrolase Gene from Pseudomonas putida Strain WW2.

During the synthesis of indigo from indole, low levels of othercompounds accumulate in the culture supernatant. One of theseby-products, isatin (indole 2,3-dione), has been found to inhibit NDOactivity in the production strain with the consequence of reducingoverall indigo yield. Furthermore, isatin is a precursor to indirubin, ared dye which may cause a slight reddish cast on materials dyed withmicrobial indigo. Methods were, therefore, sought to remove or eliminatebuildup of isatin. Accordingly, a search for an enzyme capable ofdegrading isatin was initiated. Numerous soil samples were screenedusing a nutritional selection scheme, resulting in the identification ofan organism exhibiting the ability to degrade isatin. Taxonomic studiesconsisting of Biolog®, GC-FAME, gelatin hydrolysis and phospholipase Cassays suggest the organism to be a Pseudomonas putida. The organismwas, therefore, designated Pseudomonas putida strain WW2. The enzymaticactivity was identified as a hydrolytic reaction in which isatin ishydrolyzed to isatic acid. The enzyme was, therefore, designated ‘isatinhydrolase.’

To clone the gene encoding isatin hydrolase (see FIGS. 2 and 3), totalDNA was prepared from Pseudomonas putida strain WW2 and partiallydigested with Sau3A restriction endonuclease. The partially digested DNAwas then electrophoresed in a 0.7% agarose gel for 12 hours at 100 mV.Following staining with ethidium bromide, the DNA was visualized byfluorescence and a gel slice containing DNA fragments ranging from 1 kbto 10 kb was excised. The DNA was eluted from the gel slice byelectroelution and further purified by phenol/chloroform treatment,ethanol precipitation and resuspension in TE buffer.

The isolated DNA fragments were then ligated to the expression vectorpTrc99A (commercially available from Pharmacia Biotech, Inc.; catalog#27-5007-01) which was previously linearized by digestion with BamHI anddephosphorylated with calf intestinal alkaline phosphatase. Followingligation, 5 μl of the ligation mixture was used to transform competentE. coli Sure Cells® (obtained from Stratagene, catalog #200238).Following the outgrowth of the transformation, the mixture was diluted1:1 with 2YT medium and plated on L-agar plates containing 50 μg/mlcarbenicillin and 1 mM IPTG.

Following overnight incubation of the plates at 37° C., several thousandtransformants were observed. Colonies were lysed by spraying the surfaceof the plates with a solution of 10 mg/ml lysozyme and 25 mM EDTA andincubating for 20 minutes at room temperature. Plates were then overlaidwith nitrocellulose membranes previously stained for 20 minutes in asolution consisting of 3.5 mg of 5,7-dimethylisatin in 10 ml of 50 mMTris/HCI pH 7.5. After a one hour incubation period the nitrocellulosemembranes were lifted from the plates and inspected for white clearingzones on an orange background. Such clearing zones would indicate thepresence of an isatin hydrolytic activity.

This screen yielded a positive transformant which contained a plasmidwith a 6.3 kb cloned insert (FIG. 1). This plasmid was designatedpTrc-IH. Subcloning of the isatin hydrolase gene was accomplished bydeleting segments of the 6.3 kb insert from pTrc-IH. Digestion ofpTrc-IH with SalI gave 3 fragments (1.6 kb, 2.6 kb, and 6.3 kb). Thisindicated that the cloned insert contained a minimum of two internalSalI sites. The cloning vector contains one SalI site in the polylinkerregion. When the 6.3 kb SalI fragment of pTrc-IH was isolated,re-ligated, and transformed into competent E. coli Sure Cells®(Stratagene, catalog #200238) isatin hydrolase activity was againobserved. This new plasmid was designated pTrc-IH#2, and contained theIH gene on a cloned fragment of approximately 2.3 kb (FIG. 1).

Restriction mapping of pTrc-IH#2 revealed the presence of a unique XhoIrestriction site in the cloned fragment located approximately 400 basepairs from one of its ends. This permitted further subcloning of theisatin hydrolase-containing fragments (see FIG. 2). pTrc-IH#2 wasdigested with XhoI followed by filling of overhangs with T4 DNApolymerase (creating blunt ends). The resultant linearized plasmid wasthen digested with SalI. This generated a 1.9 kb blunt-SalI fragmentwhich was purified from an agarose gel and ligated to the vector pUC18previously digested with Smal and SalI. Ligation of a filled-in XhoIoverhang to a Smal end recreates a XhoI site. E. coli strain JM101 (ATCC33876) was transformed with this ligation mixture and transformantshaving isatin hydrolase activity were identified using the screendescribed above. The plasmid isolated from the positive transformant wasconfirmed to contain the isatin hydrolase gene on the 1.9 kb XhoI-SalIfragment; this plasmid was designated pUC18-IH (FIG. 2). Furtherexperiments showed that the isatin hydrolase activity in strainscontaining pUC18-IH was inducible with IPTG, suggesting that thedirection of transcription of the isatin hydrolase gene in pUC18-IH wasthe same as the lac promoter.

The isatin hydrolase gene was further subcloned as follows (see FIG. 2).Partial DNA sequencing and restriction mapping of the 1.9 kb clonedfragment in pUC18-IH revealed the presence of a Bbsl restriction siteunique to the fragment and near its center. Approximately 1 kb of thecloned DNA could be deleted by digesting pUC18-IH with Bbsl and SalI andseparating fragments on agarose gel and isolating the large (3.6 kb)fragment. After filling in ends with T4 DNA polymerase, this fragmentwas recircularized by ligation. The ligation mixture was then used fortransforming E. coli strain JM101 (ATCC 33876) and screening for isatinhydrolase activity. A positive clone yielded a new plasmid which wasdesignated pUC-IH (FIG. 2) and was found to contain the functionalisatin hydrolase gene within a fragment of about 900 base pairs.

DNA Sequence of the Isatin Hydrolase Gene.

The entire cloned insert in pUC-IH was sequenced using the commerciallyavailable lac and reverse primers designed for use with pUC vectors(ATCC 37253). The complete nucleotide sequence of the insert is shown inFIG. 3 (Seq ID No. 1). A single open reading frame (780 bp from bp 145to bp 923 in Seq ID No. 1) within the sequenced region was identifiedusing sequence analyzer software from Genetics Computer Group, Inc. Thepredicted molecular weight (32,886) of isatin hydrolase based on thenucleotide sequence of this open reading frame was in agreement with theestimated size of isatin hydrolase protein (30,000-40,000) as determinedby gel permeation chromatography and native polyacrylamide gelelectrophoresis. N-terminal amino acid sequencing of the subsequentlypurified isatin hydrolase yielded a sequence of MTSIKLLAESLLK (Seq IDNo. 3). This sequence is in agreement with the predicted N-terminalamino acid sequence derived from the nucleotide sequence of the singleopen reading frame. These analyses indicate that there are 144 basepairs 5′ (untranslated) and 82 base pairs 3′ (untranslated) adjacent tothe open reading frame.

Construction of an Auxiliary Plasmid Containing the Isatin HydrolaseGene and the E. coli K-12 Tryptophanase Gene for Use in the IndigoProduction Strain.

The concentration of isatin has been found to be reduced in tryptophanto indigo biotransformation fermentations in the presence of isatinhydrolase when supplied to the fermentation either as a cell extract orwhen the enzyme was expressed from a plasmid. A separate benefit in therate of indigo production in shake flasks was observed whentryptophanase, the enzyme that converts tryptophan to indole and isderived from E. coli K-12, was overexpressed from a plasmid harboringthe tnaA gene. Both the isatin hydrolase and the tryptophanase geneswere, therefore, combined on a single low copy number plasmid to effectthese benefits. The construction of this plasmid is described below.

During the construction of plasmid pUC-IH, the unique SalI restrictionsite at the 3′ end of the isatin hydrolase gene was destroyed. Forconvenient subcloning of the isatin hydrolase gene from pUC-IH, a SalIsite was recreated at the 3′ end of the isatin hydrolase gene bydigesting pUC-IH with HindIII, and cloning into this site (in the properorientation) a 3.2 kb HindIII fragment containing the E. coli K-12tryptophanase (tnaA) gene (from pSUtna, see FIG. 4) [Deeley and Yanofsky(1981) “Nucleotide sequence of the structural gene for tryptophanase ofEscherichia coli K-12,” J. Bacteriol. 147:787-796]. The latter fragmentwas chosen only because it has useful polylinker sequence at one end ofthe cloned tnaA gene. The new plasmid construction was designatedpUC-IH-H (FIG. 4). This new intermediate construction allowed the isatinhydrolase gene to be conveniently removed from pUC-IH-H on a XhoI-SalIfragment of approximately 1 kb.

This fragment was cloned in both orientations into the unique SalI siteof the low copy number vector pCL1920 [C. G. Lerner and M. Inouye (1990)Nucleic Acid Research 18:4631]. The desired plasmid with the isatinhydrolase gene in the same orientation as the lac promoter and a uniqueSalI site at the 3′ end of the isatin hydrolase gene (see FIG. 5) wasisolated and designated pCL-IH-S1.

The cloned E. coli K-12 tryptophase (tnaA) gene was added to pCL-IH-S1as follows: a 1.3 kb EcoRI fragment containing the kanamycin resistancegene from pMB2190 [A. Darzins, B. Frantz, R. I. Vannags, A. M.Chakrabarty (1986) Gene 42:293-302] was inserted into the unique EcoRIsite at one end of the cloned tnaA gene in plasmid pSUtna. PlasmidpSUtna was constructed by subcloning a 3.2 kb EcoRI-BamHI fragment(containing tnaA) from plasmid pMD6 [M. C. Deeley and C. Yanofsky (1981)J. Bacteriol. 147:787-796] into plasmid pSU18 [B. Bartolome, Y. Jubete,E. Martinez, F. de la Cruz (1991) Gene 102:75-78]. To introduce anadditional SalI site adjacent to the tnaA gene, a 1.3 kb EcoRI fragmentcontaining the kanamyacin resistance gene and two flanking multiplecloning sites from pMB2190 [partial plasmid map is shown in FIG. 8; A.Darzins, B. Frantz, R. I. Vanags, A. M. Chakrabarty (1986) Gene42:293-302] was introduced into the unique EcoRI site, 3′ to cloned tnaAin plasmid pSUtna. The resultant plasmid was designated pAK1.

The tnaA gene could be excised from pAK1 as a 3.2 kb SalI fragment. Thisfragment was inserted into the unique SalI site of pCL-IH-S1 (see FIG.7). Of the two predicted orientations of the insert, the one in whichthe orientation of transcription was the same as for isatin hydrolasewas isolated and designated pCL-IST1 (FIG. 7).

Construction of the Production Organism.

To create the production organism, the compatible plasmids 911-ISP[Ensley et al. (1987) “Expression and complementation of naphthalenedioxygenase activity in Escherichia coli ” in Microbial Metabolism andthe Carbon Cycle, S. R. Hagdorn, R. S. Hanson and D. A. Kunz, eds.,Harwood Academic Publishers, New York, pp. 437-455] and pCL-IST1 (FIG.7) were introduced into the production host FM5 [Burnette et al. (1988)“Direct expression of Bordella pertussin toxin subunits to high levelsin Escherichia coli,” Bio/technology 6:699-706] by the standardtransformation procedure using FM5 cells rendered competent by calciumchloride treatment. Transformants containing both plasmids wereidentified by their resistance to ampicillin and spectinomycin conferredby plasmids 911-ISP and pCL-IST1, respectively. The presence of theseplasmids in the FM5 host was confirmed by isolation of total plasmid DNAand restriction enzyme analyses.

The production host strain, FM5, was previously described [Burnette etal. (1988) “Direct expression of Bordella pertussin toxin subunits tohigh levels in Escherichia coli” Bio/technology 6:699-706] as was theproduction plasmid, 911-ISP, and the host FM5/911-ISP [Ensley et al.(1987) “Expression and complementation of naphthalene dioxygenaseactivity in Escherichia coli” in Microbial Metabolism and the CarbonCycle, S. R. Hagdorn, R. S. Hanson and D. A. Kunz, eds., HarwoodAcademic Publishers, New York, pp. 437-455]. The cloned E. coli K-12 tnafragment encoding tryptophanase (tnaA) (3.2 kb) has been described[Deeley and Yanofsky (1981) “Nucleotide sequence of the structural genefor tryptophanase of Escherichia coli K-12,” J. Bacteriol. 147:787-796],as has been the vector pCL1920 [C. G. Lerner and M. Inouye (1990) “Lowcopy number plasmid for regulated low-level expression of cloned genesin Escherichia coli with blue/white insert screening capability,”Nucleic Acid Research 18:4631]. The only new DNA being introduced intothe indigo production strain (FM5/911-ISP, pCL-IST1) is the pCL1920vector with a 1 kb XhoI-SalI fragment containing the isatin hydrolasegene inserted along with a small amount of sequenced polylinker DNAcarried over from intermediate plasmid constructions (see FIG. 8) andthe tna DNA coding for tryptophanase.

The application of the isatin hydrolase gene to remove isatin producedas a by-product in an indigo fermentation can take on a number ofdifferent forms. We envisage the use of the gene product isatinhydrolase in the bioconversion of tryptophan to indigo (bioconversionmethod) and in the production of indigo from glucose in a single hostorganism through the intermediate synthesis of tryptophan or indole bythis organism (single host or direct method). The residence of theisatin hydrolase gene can take on several forms itself: the gene wouldbe effective when placed on the chromosome of the host, supplied on aextrachromasomal autonomously replicating DNA element, or any other formof DNA introduced into the cell and maintained for the duration of thefermentation. The regulation of gene expression may be constitutive ormay be regulated with a suitable promoter.

When chromosomally incorporated, the hydrolase gene would work inconcert with oxygenases such as NDO (U.S. Pat. No. 4,520,103 and U.S.Pat. No. 5,173,425), TMO (U.S. Pat. No. 5,017,495) or an oxidase asexemplified by the oxidase from Rhodococcus [S. Hart, K. R. Koch and D.R. Woods (1992) “Identification of indigo-related pigments produced byEscherichia coli containing a cloned Rhodococcus gene,” J. Gen.Microbiology 138:211-216]. Single component monooxygenases capable ofproviding indoxyl either directly or indirectly are also candidates forapplication with isatin hydrolase. These oxygenases/oxidases orcomponents of them could be encoded either on the chromosome of the hostor be introduced on extrachromosomal elements. The genes for theoxidative enzymes could be expressed constitutively or be regulated withappropriate promoters. The isatin hydrolase gene could reside on thesame DNA element as the gene encoding the oxidizing enzyme. Expressionof the genes for all activities may be regulated independently or may beregulated in concert.

In the direct method, i.e., the single host method, the isatin hydrolasegene is expected to be coexpressed along with genes of the aromaticamino acid pathway which could be under endogenous regulation orartificially regulated by engineered mutations which remove feed-backinhibition and/or cause the genes to be overexpressed. This includesaromatic amino acid pathway genes amplified by virtue of being encodedon multi-copy number plasmids from which the appropriate genes areexpressed from constitutive promoters or from regulated promoters. Allgenes could be regulated by different promoters or any combination ofpromoters.

The aromatic amino acid pathway genes could also be located solely onthe chromosome or there could be any combination of chromosomallyencoded aromatic amino acid pathway genes operating from plasmids.

The general recombinant DNA techniques used in the present invention,such as DNA isolation and purification, cleavage of DNA with restrictionenzymes, construction of recombinant plasmids, introduction of DNA intomicroorganisms, and site directed mutagenesis, are described in manypublications, including Manniatis et al., Molecular Cloning—A LaboratoryManual, Cold Springs Harbor Laboratory (1982) and Current Protocols inMolecular Biology, edited by Ausubel et al., Greene PublishingAssociates and Wiley Interscience (1987).

II. Other Approaches to Isatin Removal

Ways to implement the adsorption or chemical conversion to anon-inhibitory compound in a fermentation are known to those skilled inthe art. These may include, but are not limited to, the following.

Removal of Cell-Free Broth for Treatment.

Cell-free broth can be removed from the fermentor by means of aseparation device, such as a cross-flow filtration membrane or acentrifuge. The cell-free broth could then be treated by means shown inExamples 1 or 2. A heat exchange loop could be used to heat, and cool ifnecessary, the broth. Base for pH control could be added in the loop toincrease alkalinity. In one embodiment of the present invention theisatin by-product is removed by treating the fermentation broth byelevating the pH to at least about 11 (by adding an appropriate base)and elevating the temperature to about 50-70° C. This treatment mayoccur over a period of time sufficient for the orange color of the brothto dissipate to a pale yellow (<12 hours, preferably about 2-5 hours orless). Alternatively, the broth could be passed over activated carbon oranother adsorbent to remove the isatin [Freeman et al. (1993) “in situproduct removal as a tool for bioprocessing,” Bio/technology11:1007-1012].

Addition of Adsorbent Directly to Bioconversion.

Adsorbent could be added to the bioconversion to adsorb isatin.Activated carbon or another type of adsorbent could be added to thebroth and then separated from the indigo after the fermentation.

Hydrolysis of Isatin by General Base Catalysis.

The concentration of isatin could be lowered by the addition of elevatedphosphate, which acts as a general base catalyst for the hydrolysis ofisatin, to a fermentation. General base catalysis is shown in Example 4.

EXAMPLE 1 Removing Isatin From a System Increases Indigo Production.Alleviation of Inhibition of Indigo Formation by Treatment with Heat andElevated pH

Two fermentations were performed with a strain capable of convertingtryptophan to indigo [FM5(911-ISP, pCL-ISP#14)]. One was fed tryptophanand produced indigo while the control tank was not fed tryptophan anddid not make any indigo. Broth was taken from each tank at 20 hoursfermentation time and centrifuged to remove indigo and cells. Glucosewas added to the supernatants to 10 g/L. For additional controls buffersamples containing 200 mM K⁺ phosphate at pH 7.0 with 10 g/L glucose and±5 mM isatin were prepared. To an aliquot of each broth and each buffer,45% KOH was added to raise the pH to about 11.0. The solutions wereplaced on a hot plate and heated at about 50-70° C. until the orangecolor of the isatin solution changed to a pale yellow (about 2-5 hours).This color change, as determined in previous spectral experiments,indicates that isatin is hydrolyzed to isatic acid as shown in Scheme 1.At this time, all samples were cooled and the pH was readjusted to 7.0with 85% phosphoric acid. isatin isatic acid

Cells were taken at 21 hours from the fermentor in which no tryptophanwas fed and centrifuged. After resuspension in 200 mM K⁺ phosphate at pH7.0 with 10 g/L glucose buffer at high cell density, equal aliquots ofcells to give a final A⁶⁶⁰ of 2 were placed into 25 mL of the pH andheat treated buffers and broth contained in a 250 mL baffled flasks,respectively (carried out in duplicate). The flasks were shaken for 30minutes, at which time an initial indigo measurement was made (bydissolving 1 part culture in 10 parts or greater of DMF and measuringA⁶⁶⁰) and indole was added to a concentration of 250 mg/L. Indigoproduction was measured spectrophotometrically at A⁶¹⁰ at the end of onehour and expressed as gram indigo per gram dry cell weight per hour.Results (for duplicate runs) are recorded in Table 1, comparing treatedand untreated broth for the production of indigo.

TABLE 1 Indigo Production in pH and Heat Treated Fermentation Broth andBuffer as Determined by Shake Flask Assay Heat and pH Treatment MediumTreated Untreated Non-Trp Fed Broth 0.400 0.446 0.442 0.442 Trp FedBroth 0.314 0.347 0.0843 0.0797 Buffer With Isatin 0.327 0.327 0.06270.0764 Buffer 0.285 0.307 0.311 0.321

Results show that isatin or broth from an indigo producing bioconversioncan reduce further indigo production. Treatment of broth or buffer tohydrolyze isatin to isatic acid relieves such inhibition.

EXAMPLE 2

Increase in Indigo Production By Treating Spent Broth From aBioconversion With Activated Carbon

Broth was collected from a bioconversion as in Example 1. Activatedcarbon was used to treat one aliquot of broth, with an aliquot of brothwith no treatment serving as control. Indigo production rates,determined as in Example 1, are shown in Table 2. Results are shown asduplicates of the experiment.

TABLE 2 Indigo Production in Activated Charcoal Treated FermentationBroth as Determined by Shake Flask Assay Indigo Production Broth(g/gDW/hr) Non-Carbon Treated 0.057 0.048 Carbon Treated 0.115 0.122

These data show that treatment with carbon can relieve inhibition ofindigo production. Release of inhibition was attributed to the removalof isatin by activated charcoal.

EXAMPLE 3 Isatin is a Redox Cycler with Purified NDO

Recombinant NDO was purified, by published procedure [Haigler and Gibson(1990) “Purification and properties of NADH-ferredoxin_(nap) reductase,a component of naphthalene dioxygenase from Pseudomonas sp. Strain NCIB9816,” J. Bacteriol. 172:457-464; Ensley and Gibson (1983) “Naphthalenedioxygenase: purification and properties of a terminal oxygenasecomponent,” J. Bacteriol. 155:505-511; Haigler and Gibson (1990)“Purification and properties of ferredoxin_(nap), a component ofnaphthalene dioxygenase from Pseudomonas sp. Strain NCIB 9816,” J.Bacteriol. 172:465-468] from an E. coli strain overexpressing ferredoxinreductase (Rd), ferredoxin (Fd) and terminal oxygenase (ISP). The NDOoperon had been placed under the phoA promoter. Purified components werestored at −80° C. until used. Isatin was established as an electronacceptor for ferredoxin reductase and ferredoxin by spectrallymonitoring NADH oxidation at 340 nm in the presence of isatin. Assayswere conducted as follows: 0.97 ml 50 mM Tris/HCI pH 7.5, 100 uM NADH,redox enzyme components and isatin. In some experiments NADH was alsomeasured in the presence of the NDO substrate(R)-1,2,3,4-tetrahydro-1-naphthalene [(R)-THN]. In the absence of isatinor substrate no NADH oxidation was observed. Isatin did not changeconcentration in any assay as determined by monitoring its spectrum.Table 3 below summarizes results.

TABLE 3 Summary to Experiments Demonstrating Redox Cycling by IsatinSubstrates NADH (R)-THN = 1 mM Oxidized Enzyme Components [NADH], mMIsatin = 250 uM nmol/min/ml Present 0.100 (R)-THN 9.0 ISP, Fd, Rd 0.100(R)-THN + isatin 15.0 ISP, Fd, Rd 0.100 isatin 10.9 ISP, Fd, Rd 0.100isatin 12.3 Fd, Rd 0.100 isatin 3.4 Rd 0.140 isatin 13.1 ISP, Fd*, Rd0.100 isatin 12.5 ISP, Fd*, Rd Note: [ISP] = 1 uM, [Rd] = 60 nM, [Fd] =0.94 uM except when indicated by *, where [Fd]0 was 0.67 uM. Assay wasnot optimized for these experiments. In the absence of enzyme componentsno NADH oxidation was observed.

Results clearly show that isatin mediates the oxidation of NADH. Sinceisatin does not undergo any chemical change it acts as a redox cyclerwith the Rd and Fd components.

EXAMPLE 4 Demonstration of General Base Catalyzed Hydrolysis of Isatin

Hydrolysis of isatin by general base catalysis was demonstrated atseveral different concentrations of KPO₄ buffer pH 7 and 37° C.Hydrolysis was followed spectroscopically by monitoring disappearance ofisatin and appearance of isatic acid at 302 and 368 nM, respectively.The starting concentration of isatin was 250 uM. The following tablesummarizes rates and demonstrates dependence of rate on bufferconcentration. The table also shows the rate of isatin hydrolysis for anumber of other anion and buffers, all at pH 7 and 37° C. Rates areexpressed as half-life (τ½). Table 4 summarizes isatin hydrolysis ratesin buffers.

TABLE 4 Summary of Data Demonstrating Hydrolysis of Isatin in VariousBuffers Buffer Concentration (mM) τ½ Tris-HCl 50 hours Tris-AcOH 50hours Tris-HCl + 50 hours NaCl 100 Tris-HCl + 50 hours KCl 100 Kphosphate 25 151 min  50 85 min 100 45 min 200 22 min Na phosphate 10035 min K pyrophosphate 100 35 min Na triphosphate 100 70 min NaCarbonate 100 80 min

EXAMPLE 5 Isolation of Organism Producing Isatin Hydrolase

The isatin hydrolase-producing organism was isolated from a soil samplecollected at a creosote plant in Terre Haute, Ind. Isolation wasaccomplished by an enrichment protocol using minimal salt medium[Stanier et al. (1957) J. Cell Comp. Phys. 49:25] containing 1 g of thesoil sample and 1.7 mM indole as the sole carbon and energy source.After three serial passages of liquid enrichment cultures the organismwas purified by plating liquid culture on 1.5% minimal salt medium agarplates with indole as the sole carbon source, followed by plating agrowing colony on an LA plate and finally streaking a 1.5% minimal saltmedium plate containing indole with cells derived from a single colonyfrom the LA plate. Enrichment and purification were carried out at 30°C. Whole cells derived from a single colony and cultured in the originalminimal salt medium with indole exhibited isatin hydrolase activity.This assay is described in Example 6. This colony was designated strainWW2.

EXAMPLE 6 Demonstration of Isatin Hydrolase Activity of Whole Cells ofOrganism (WW2) Isolated in Example 5

Cells grown in a minimal salt medium containing 1.7 mM indole werecentrifuged. The cell pellet was resuspended in 50 mM Tris-HCI, pH 7.5to an OD⁶⁰⁰ of 2. To an aliquot of cell suspension, isatin was added toa concentration of 200 μM. The orange color disappeared in <30s. Cellswere removed from the sample by centrifugation and the spectrum of thesupernatant was recorded. This spectrum was identical to authenticisatic acid. HPLC analysis confirmed the identity of the product.

EXAMPLE 7 Demonstration of Isatin Hydrolase Activity in Cell Free Assayof WW2 and Apparent Native Molecular Size

A 1:4 cell homogenate in 50 mM Tris-HCI, pH 7.5, of the organismisolated in Example 5 and grown either in LB medium containing 1.7 mMindole or in a minimal salt medium containing 1.7 mM indole was preparedby disrupting the cells in a French pressure cell. The homogenate wasassayed directly or the supernatant and the pellet were assayed after100,000 g centrifugation of the homogenates. Isatin hydrolase activitywas found in the whole homogenates and in the high speed supernatants.Less than 5% of activity was detected in the high speed pellets.Equivalent activity was detected whether the cells were grown in rich orminimal medium. Cells grown in rich medium in the absence of indole had20-fold reduced activity, indicating that indole induces the enzymeactivity.

Apparent native molecular size was determined as follows. The crudehomogenate from above was fractionated on DEAE cellulose with a 0 to 500mM NaCL gradient in 50 mM Tris/HCI, pH 7.5. Enzymatic activity eluted at˜175 mM NaCl, however, 85% of the enzymatic activity was lost duringthis single step. Pooled and dialyzed fractions were furtherfractionated by a 30% to 40% NH₄SO₄ precipitation. Further losses inactivity to about 10% of original activity resulted. Further treatmentof the sample with TSK Q ion exchange chromatography yielded 1.4% oforiginal activity on elution with a 0 to 500 mM NaCl gradient in thesame buffer. This remaining activity was finally applied to a SepharoseS-100 gel filtration column. Enzymatic activity eluted corresponding toan apparent molecular weight of about 30 to 40,000 Da. Steps describedabove were monitored by spectrophotometric enzymatic assay. Eitherdisappearance of isatin or appearance of isatic acid could be monitoredat 302 or 368 nM, respectively, in 50 mM Tris/HCI, pH 7.5. Activitycould also be detected on a 7.5% native acrylamide gel by overlaying thedeveloped gel with a nitrocellulose membrane previously soaked in5,7-dimethylisatin as described in Example 14. Enzyme was located by theappearance of a white spot on the peach colored membrane, the colorhaving been imparted by incubation of the membrane with a 3.5 mg/10 ml50 mM Tris HCI pH 7.5, 5,7-dimethylisatin solution for 20 min.

EXAMPLE 8 Increase In Indigo Production by Treating Spent Broth From aBioconversion with Extract From WW2, Producing Isatin Hydrolase

Broth was collected as in Example 1. A cytosolic cell extract fromstrain WW2, prepared by breaking cells with a French pressure cell andcentrifugation at 100,000 g, was added to the broth and E. coli hostcells and contacted for one hour prior to indole addition. Indigoproduction rate was measured and is shown in Table 5.

TABLE 5 Indigo Production in WW2 Cell Extract Treated Fermentation Brothas Determined by Shake Flask Assay Indigo Production Broth (g/gDW/hr)Non-Extract 0.071 Treated Extract Treated 0.234

These data show that treatment with an enzyme capable of convertingisatin to isatic acid relieves inhibition of indigo production.

EXAMPLE 9 Demonstration of Reduction of Isatin Concentration During aBioconversion of Tryptophan to Indigo in the Presence of a CytosolicExtract from Pseudomonas putida WW2

Cells of Pseudomonas putida strain WW2 were grown in a 10 L fermenter ina minimal salt medium with the addition of indole. Cell OD 600 of 4 wasattained. Cells were harvested and an extract was prepared by breakingcells as a 1:1 suspension with the aid of a French press andcentrifugation at 100,000 g. Beginning at 16 hours, this extract wasadded in 50 mL aliquots at 30 min. intervals for 4.5 hours to abioconversion of tryptophan to indigo by FM5/911-ISP, pCL-ISP#14. Withinone hour the isatin concentration had dropped from 0.4 mM to 0.06 mM.Over the same time interval the isatin concentration in a control tankrose to 0.95 mM, a level of isatin known to be inhibitory to NDOactivity. The hydrolysis product isatic acid rose from 0.16 mM to 1.64mM in the experimental tank, while in the control tank its concentrationnever exceeded 0.5 mM. This experiment showed that isatin hydrolase cansignificantly reduce the levels of isatin in broth during afermentation. This experiment also suggests that isatin may be convertedto some other product in the absence of isatin hydrolase since the sumof isatin plus isatic acid at the end of the fermentation is >7 mM whilein the control tank this sum was <2 mM. This finding is consistent withthe observation of elevated levels of indirubin in fermentations whereisatin hydrolase is absent. Indirubin can form from the reaction ofisatin with indoxyl, the precursor to indigo.

EXAMPLE 10 Taxonomic Classification of Organism Designated WW2

The organism isolated in Example 5 was typed by GC-FAME and Biolog®.These methods suggested the organism to be Pseudomonas marginalis orPseudomonas putida type A 1, respectively. Assays for phospholipase Cactivity [R. M. Berka, G. L. Gray and M. L. Vasil (1981) “Studies ofphospholipase C (heat-labile homolysin in Pseudomonas aeruginosa),”Infection and Immunity 34:1071-1074] and the gelatin liquefactionactivity [R. N. Krieg, ed., (1984) Bergey's Manual of SystematicBacteriology, vol. 1 (J. G. Holt, series ed.), Williams and Wilkins,Baltimore, pp. 163-165] suggest the species to be P. putida. GC-FAME andBiolog® tests were done by Mirobe Inotech Laboratories, Inc. The newdesignation for WW2 is, therefore, Pseudomonas putida WW2.

EXAMPLE 11 Isolation of DNA Fragment Containing Isatin HydrolaseActivity

Total DNA was isolated from the organism described in Example 5 by themethod of Harwood and Cutting [(1990) Molecular Biological Methods forBacillus, John Wiley, New York, pp. 140-145]. All subsequent DNAmanipulations were carried out by standard protocols found in Sambrooket al. [(1989) Molecular Cloning—A Laboratory Manual, 2nd ed., ColdSprings Harbor Laboratory] or as suggested by the manufacturers of kits.DNA was partially digested using 0.25 units of Sau3A restrictionendonuclease (NEB) per 10 μg of DNA for one hour at 37° C. Digested DNAwas fractionated on agarose gel and fragments of 1 to 10 kb wereisolated by electroelution. A plasmid library was constructed using thepTrc99A expression vector (Pharmacia, catalog #27-5007). The ligationmixture was transformed into competent Sure Cells® (Stratagene, catalog#200238) which were subsequently plated onto 40,15 cm 2YT platescontaining 50 μg of carbenicillin, and 50 μg of indole per ml, and 1 mMIPTG. After overnight growth at 37° C., colonies were lysed by lightlyspraying with a solution containing 10 mg/mL of lysozyme and 25 mM EDTAand incubating at room temperature for 20 min. The partially lysed cellswere then overlaid with a nitrocellulose filter that had beenimpregnated for 20 to 30 min. in 50 mM Tris-HCI, pH 7.5, and 2 mM5,7-dimethylisatin (5,7-dimethylisatin was added from a 20 mM stocksolution made up in ethanol). Positive clones were identified as adecolorized dot in a peach colored background. A total of 3 positiveclones out of 3,000 were identified. Picked colonies were purified bytwo rounds of colony purification on solid LB plates. After isatinhydrolase activity was demonstrated as described in Example 12 below,restriction analysis of the cloned DNA showed it to be about 6.8 kb insize. Subcloning yielded a 2.3 kb and subsequently a 1 kb fragmentexhibiting isatin hydrolase activity when assayed in whole cell assaydescribed in Example 12. The complete nucleotide sequence of the 1 kbfragment was subsequently determined and is shown in FIG. 3 (Seq ID No.1). The amino acid sequence was deduced as shown in Seq ID No. 2.

EXAMPLE 12 Demonstration of Isatin Hydrolase Activity in E. coli CloningOrganism

Whole cells of positive clones identified in Example 11 were assayed forisatin hydrolase activity similarly as in Example 6, except that themedium was LB containing carbenicillin. IPTG was not required forinduction of cells containing the full length fragment or the 2.3 kbfragments, suggesting that these isolated DNA fragments contained anendogenous promoter. The subcloned 1 kb fragment, however, required IPTGto express maximal activity.

EXAMPLE 13 Construction of Plasmid pCL-IST1

As is shown in FIG. 7, construction of this plasmid was accomplished bydigestion of plasmid pAK1 with SalI, isolation of the 3.2 kb fragmentcontaining the tnaA structural gene, by agarose gel electrophoresis, andligation of this fragment into plasmid pCL-IH-S1 after its linearizationwith SalI.

EXAMPLE 14 Construction of Plasmid pCL-IHA

As is shown in FIG. 8, this plasmid was constructed in two steps fromplasmids pTrc-IH#2, pMB2190 and pCL1920. Digestion of plasmids pMB2190[Darzins et al. (1986) Gene 42:293-302] and pTrc-IH#2 with EcoRI,followed by ligation and transformation, allowed the isolation of acolony resistant to ampicillin and kanamycin which bore the plasmidpTrc-IH#2-kan. This cloning step allowed for the isolation of a 2.5 kbSalI fragment from pTrc-IH#2-kan containing the isatin hydrolase gene.This fragment was then cloned into the unique SalI site of pCL1920vector to yield pCL-IHA as one of the products.

EXAMPLE 15 Absence of Inhibition of Indigo Production by FermentationBroth From a Strain Containing the 2.3 kb Fragment Harboring the IsatinHydrolase Gene

Transformation of Production Hosts

Transformation of E. coli strains was carried out by the use of theCaCl₂ method for rendering cells competent as described in Manniatis etal., Molecular Cloning—A Laboratory Manual, Cold Springs HarborLaboratory, p. 250 (1982).

Bioconversions making indigo were performed with FM5 (911-ISP) and FM5(911-ISP,pCL-IHA) (FIG. 8). The first strain was the control strain notcontaining the gene for isatin hydrolase, while the second straincontained a 2.3 kb DNA fragment harboring the isatin hydrolase gene.Broth and cells were collected from each fermentation and broth wassupplemented with glucose as in Example 1. The cells were thenresuspended either in the glucose supplemented broths, or buffer forindigo shake flask assays as in Example

TABLE 6 Effect of Isatin Hydrolase Gene on Indigo Production in ShakeFlask Assays Indigo Production Medium Strain (g/gDW/hr) BrothFM5(911-ISP) 0.0416 Buffer FM5(911-ISP) 0.115 Broth FM5(911-ISP,pCL-IHA)0.286 Buffer FM5(911-ISP,pCL-IHA) 0.264

Data comparing buffer and broth show that broth from a strain harboringisatin hydrolase is not inhibitory to indigo production, while that froma broth without isatin hydrolase is inhibitory.

EXAMPLE 16 DNA Sequence of the Isatin Hydrolase Gene

DNA sequence of the 1 kb fragment isolated in Example 11 was determinedby a modification of the dideoxy chain termination method [Sanger et al.(1977) Proc. Natl. Acad. Sci. USA 74:5463-5467]. An open reading frame(ORF) consisting of 780 bp was identified. There were 144 bp of5′-untranslated and 82 bp of 3′-untranslated DNA, respectively, giving atotal of 1006 bp for the cloned DNA. The sequence data is shown in FIG.3 (Seq ID No. 1).

EXAMPLE 17 Purification of Isatin Hydrolase

Partial purification of the enzyme coded for by the 1 kb DNA fragmenthas been achieved. This was accomplished by modifying the 1 kb fragmentby joining 6 histidine codons in frame to the 5′ end of the ORFdescribed in Example 14 and growing cells transformed with thisconstruct under IPTG induced conditions. Cells suspended in 50 mMTris-HCI, pH 7.5, at one part cell pellet and one part buffer andruptured in a French pressure cell. The homogenate was further dilutedwith an equal volume of the same buffer and centrifuged at 100,000 g.The high speed supernatant was passed over a Ni-NTA resin columnaccording to the manufacturer's recommendations (QIAGEN, Inc., Calif.).Enzymatic activity bound to the column and purification was achieved bywashing the column with 50 mM Na phosphate, 300 mM NaCl, 10% glycerol atpH 6.0 (wash buffer) until the A₂₈₀ dropped below 0.04 absorbance units.The column was then eluted with a gradient of 0-0.5 M imidazole in 50 mLof wash buffer. Fractions containing enzymatic activity, as detected byisatin hydrolysis, were electrophoresed on SDS-PAGE [U.K. Laemmli (1970)Nature 277:680-685]. The silver stained gel showed a major band at33,000 Da and only traces of other components in the most pure fraction.These results are in agreement with the molecular weight determinationof the protein from the native organism described in Example 7. In thiscase, gel permeation chromatography gave an estimate of 30 to 40 kDa forthe protein exhibiting isatin hydrolase activity. These indicate thatisatin hydrolase is a monomer in its native state.

EXAMPLE 18 Demonstration of Diminution of Indirubin Levels Due to IsatinHydrolase in Indigo Fermentations

Fermentations in 14 L fermenters were carried out as usual, either withan indigo-producing strain containing the gene for isatin hydrolase orwith this gene absent. Samples were taken as a function of fermentationtime and analyzed as follows: 1 ml fermentation broth was centrifugedfor 15 min. at 12,000 g, supernatant was removed and the pelletresuspended in THF/DMSO (1/1; 1 ml) and vortex for 1 min. The mixturewas recentrifuged at 12,000 g. The supernatant was used for HPLCanalysis as described in Example 20.

TABLE 7 Results for Indirubin Analyses as a Function of Presence andAbsence of Isatin Hydrolase and Fermentation Time Run X-BLU-22720 + RunX-BLU-22724 − time hydrolase hydrolase [h] [g indirubin/l] [gindirubin/l] 4 0 0 8 0 0 12 0 0 16 0.0 0.0 20 0.032 0.044 28 0.011 0.07232 0.052 0.11 36 0.059 0.155 40 0.033 0.154 44 0.064 0.250 48 0.0940.192

Additional endpoint analyses (at about 48 h, the usual termination offermentations) were carried out on a number of fermenters by the samemethod. Results for these are reported in the table below.

TABLE 8 (Indirubin HPLC Data for Runs 22741, 22743-46 (with IH) and22598 (without IH) Fermenter Run ID# Isatin Hydrolase Indirubin [g/lprep.] 22741 + 0.27 22743 + 0.09 22744 + 0.10 22745 + 0.06 22746 + 0.1722717 + 0.09 22756 + 0.09 22757 + 0.13 22759 + 0.14 22760 + 0.13 22761 +0.27 22598 − 1.13 cone 1 − 0.91

Conclusion: These experiments show that there is a twofold or greaterreduction in levels of indirubin due to the presence of adding theisatin hydrolase gene to the indigo producing strain.

EXAMPLE 19 Demonstration of Diminution of Indirubin Levels Due to IsatinHydrolase in Denim Dyed with Indigo from E. Coli Fermentation Process

Indirubin levels of indigo dyed denim correlate with a red cast of thedyed cloth and resistance to bleaching of indigo dyed denim to achieve a‘bleached out’ fashion look. Experiments below demonstrate higher levelsof indirubin in denim dyed with indigo from The E. coli fermentationprocess without isatin hydrolase than with isatin hydrolase. Indirubinlevels in denim samples were determined as follows: 2 grams of denimwere Extracted with 150 ml ethanol in a soxlet apparatus for 1.5 hours.Extracts were dried down in a rotary evaporator and brought up in 1 mlTHF:DMSO solvent for HPLC analysis according to Example 20.

TABLE 9 Indirubin Content to Denim Dyed with Indigo Produced by HostCells with and Without Isatin Hydrolase Denim Indirubin Sample SampleIsatin Indirubin Level Concentration ID Description Hydrolase in IndigoDye (mg/g denim) NI94241 fermentation − 0.91 mg/mL 0.061 indigo 20%paste NI11095 fermentation + 0.46 mg/mL 0.011 indigo 20% paste BC034chemical N/A 0.03 mg/mL below detection indigo 20% paste limit

The results show that indirubin levels are lower on denim dyed withindigo containing lower levels of indirubin. The correlation is notlinear and the factors controlling the relationship are not fullyunderstood. Additional qualitative observations (by experts in thedyeing industry) are in agreement with quantitative observations.

EXAMPLE 20

Demonstration of Effect of Indirubin on Bleach Down of Denim Dyed withIndigo Typical Procedure for Dyeing of Denim with Indigo 1. ClothHydration (predyeing conditioning) A. Cut 9″ by 15″ sections (swatches)of bull denim (two per dyebath) B. Soak in hot tap water overnight(minimum of 1 hr) 2. Dyebath Preparation A. Make stock mix using 60 g20% paste. While stirring on hot plate add: 74.9 g DI H2O 23.0 g 50%NaOH 60.0 g 20% indigo paste (with 5% NaOH) Heat to ˜70° C. Add 8.16 gsodium dithionite Stir to dissolve then remove from heat. B. Makebalance solution (use container in which dyeing will be performed):4,383 g deionized water 25.3 g 50% NaOH 15.4 g sodium dithionite Add138.4 g of stock mix. Stir to mix and then stop mixing. 3. DyeingProcedure A. Water soaked swatches are pressed between laundry-typewringer to remove excess water and assure uniform water content of allswatches prior to dyeing. B. Swatches are dipped according to followingprotocol. Submerge swatch in dyebath for 20 seconds, remove excess dyebath liquor by passing cloth through wringer, expose cloth to ambientair (skying) for approximately 3 minutes for oxidation of leucoindigo toindigo between dips. (Table of dipping schedule indicating beginning andend of dips 1 through 5 in minutes and seconds; wringing and skying iscarried out between dips.) Dip 1  0:30-0:50 Dip 2  4:00-4:20 Dip 3 7:30-7:50 Dip 4 11:00-11:20 Dip 5 14:30-14:50 Following last dip, passthrough wringer and sky for 5 minutes. C. Rinse by dipping in cold waterfollowed by dipping in hot water. Pass cloth through wringer and dry. D.When required, cut each swatch into fifteen (15) 3″ by 3″ squares.Bleaching Indigo-Dyed Swatches with Sodium Hydrochloride (SimulatingBleaching Conditions Used to Create Fashion Look) Bleaching Procedure A.Bleach bath Preparation 2.6 g sodium hypochloride to 1 L deionized water(hypochloride is added from a 5.25% stock solution) Heat to 50° C. B.Capacity of Bath No more than 1:50 ratio of cloth to bleach solution(200 g dry cloth per 10 L bleach solution) (Bleaching is carried out asspecified in appropriate Examples) C. Addition Times Separate the 15small swatches into five groups of three. Prewet in DI water for minimumof five minutes. Four of these groups will be added to the bleachsolution. Time of addition will be 0, 10, 20 and 25 minutes for time inbleach of 30, 20, 10 and 5 minutes. D. Quenching and Washing Removeswatches and place in cold water Quickly rinse and add to ˜2 L of 10 g/Lsodium bisulfite Rinse in water and add ˜2 L of 10 g/L Tide w/o bleachRinse thoroughly Dry swatches thoroughly E. Read L-values on Hunter Labcolor meter Indirubin was added at 10 mg and 70 mg indirubin/g of indigoto commercial chemical indigo paste. The ‘doped’ dye was mixed well anddenim cloth was dyed according to the dyeing and bleaching protocolsdescribed in this Example 20. Color values were read as provided below.The plotted data is shown in FIG. 9.

Evaluation of Efficiency of Bleaching of Indigo Dyed-Denim with SodiumHypochloride

Effect of bleaching was measured as L-value with a Hunter Lab colormeter (Hunter Associates Laboratory, Inc., Reston, Va.). L-valuedesignates the ‘lightness’ of a sample, with 0 representing black and100 representing pure white. Results are expressed in delta L-value.

Determination of Indirubin Concentration by HPLC

The method is as follows: column, 250×4.6 mm, RP-18, 5 μm; detection at254 nm; solvent system used, solvent A—90% water, 10% acetonitrile, 2g/L tetrabutylammoniumbromide, solvent B—90% acetonitrile, 10% water, 2g/L tetrabutylammoniumbromide.

Gradient program:

flow % A % B time (ml/min) 65 35 0 0.7 65 35 2 0.7 35 65 50 0.7 65 35 510.7 65 35 55 0.1

Retention time of indirubin in this system is 31.8 min., and indigoelutes at 27.5 min.

The data in FIG. 9 shows that at elevated levels of indirubin, bleachingis inhibited. Although the difference seems small at 30 min., theskilled artisan viewing cloth dyed with indigo, with or withoutindirubin, recognizes the difference as being significant. While thesample without indirubin and with 10 mg indirubin added metspecifications, the sample with 70 mg indirubin did not.

3 1006 base pairs nucleic acid single linear DNA (genomic) not provided1 CGAGGACTGG AGAACAGAGA GCTATAAGAT TTATGCAATT TACCCGTCAA GAAAAAACAT 60GAACCCAGCA CTTCGGGTTT TTCTGGACTA TATGTACCTT CACCTCCCGC ATCAGATTTC 120CGGGACTTCT TTCGAGGAAA ACTCATGACC AGCATTAAAC TCCTTGCAGA GAGTCTGCTC 180AAAGACAAAA TAAAGATCGT CGATCTATCG CACACCTTGA GATCCGAATT TCCGACACTG 240ACATTACCTC CTCAGTTTGG GCAAACCTGG GCGTTCAAGA AGGAGGAAAT ATCGCGCTAC 300GACGACCGTG GGCCCGCTTG GTACTGGAAC AACTTTTCCT GCGGCGAACA CACTGGTACT 360CACTTTGATG CCCCAGTCCA TTGGGTCACA GGCGAATCCG TGCCTGAGAA CTCAGTAGAT 420CGTATTGACC CACAGCGCTT TATGGCACCG GCAGTAGTGA TTGATGCCTC TAAAGAGGTA 480CTAGAAAATC CGGACTGGGT TCTAGAGCCA GAATTTATCC AGGAGTGGGA GAAACTGCAT 540GGCCGGATCG AAGCCGGTTC CTGGTTTCTA CTCCGGACAG ATTGGTCGAA GAAAATCAAT 600AACCCGCTTG AGTTTGCTAA CCTGATAGAC GGCGCACCTC ACACGCCAGG CCCAAGCCAG 660CGTACAGTTG AATGGCTTAT CGCCGAACGT GATGTCGTGG GCTTTGGGGT TGAGACGATC 720AATATTGATG CGGGCCTTTC AGGCCGCTGG GAAGTTCCAT ACCCTTGCCA CAACAAGATG 780CTGGGAGCAG GACGATTCGG GCTGCAGTGC TTGAACAATC TTGACCTGTT ACCACCAACA 840GGAGCAGTAA TCATCTCCGC TCCACTGAAG ATCGAAGATG GCTCAGGCAG CCCGCTGCGG 900GTACTGGCTA TTTTTGATCG AGAATAACTG AGAGTACCCT GGGGCCGATA GACTCATCGG 960CCCCAAGTGA GTGTTCTCTA CTCGTAGTAG AAGCGAAGAC CAACTT 1006 260 amino acidsamino acid single linear protein not provided 2 Met Thr Ser Ile Lys LeuLeu Ala Glu Ser Leu Leu Lys Asp Lys Ile 1 5 10 15 Lys Ile Val Asp LeuSer His Thr Leu Arg Ser Glu Phe Pro Thr Leu 20 25 30 Thr Leu Pro Pro GlnPhe Gly Gln Thr Trp Ala Phe Lys Lys Glu Glu 35 40 45 Ile Ser Arg Tyr AspAsp Arg Gly Pro Ala Trp Tyr Trp Asn Asn Phe 50 55 60 Ser Cys Gly Glu HisThr Gly Thr His Phe Asp Ala Pro Val His Trp 65 70 75 80 Val Thr Gly GluSer Val Pro Glu Asn Ser Val Asp Arg Ile Asp Pro 85 90 95 Gln Arg Phe MetAla Pro Ala Val Val Ile Asp Ala Ser Lys Glu Val 100 105 110 Leu Glu AsnPro Asp Trp Val Leu Glu Pro Glu Phe Ile Gln Glu Trp 115 120 125 Glu LysLeu His Gly Arg Ile Glu Ala Gly Ser Trp Phe Leu Leu Arg 130 135 140 ThrAsp Trp Ser Lys Lys Ile Asn Asn Pro Leu Glu Phe Ala Asn Leu 145 150 155160 Ile Asp Gly Ala Pro His Thr Pro Gly Pro Ser Gln Arg Thr Val Glu 165170 175 Trp Leu Ile Ala Glu Arg Asp Val Val Gly Phe Gly Val Glu Thr Ile180 185 190 Asn Ile Asp Ala Gly Leu Ser Gly Arg Trp Glu Val Pro Tyr ProCys 195 200 205 His Asn Lys Met Leu Gly Ala Gly Arg Phe Gly Leu Gln CysLeu Asn 210 215 220 Asn Leu Asp Leu Leu Pro Pro Thr Gly Ala Val Ile IleSer Ala Pro 225 230 235 240 Leu Lys Ile Glu Asp Gly Ser Gly Ser Pro LeuArg Val Leu Ala Ile 245 250 255 Phe Asp Arg Glu 260 13 amino acids aminoacid single linear protein not provided 3 Met Thr Ser Ile Lys Leu LeuAla Glu Ser Leu Leu Lys 1 5 10

What is claimed is:
 1. An improved process for the fermentativeproduction of indigo, the process comprising culturing in a fermentationbroth under appropriate conditions to produce indole or indoxyl, asuitable host microorganism capable of producing indole or indoxyl, theimprovement comprising treating the fermentation broth of saidmicroorganism under appropriate conditions in a manner to remove isatinor indirubin present in said fermentation broth, wherein the manner ofremoving isatin or indirubin is by contacting the broth with anisatin-removing enzyme, said enzyme comprising the amino acid sequenceshown in SEQ I D NO:2.